Go is a guanine nucleotide-binding protein that is involved in the regulation of signal transduction in animal cells. It is composed of alpha, beta, and gamma subunits, with the a subunits possessing a guanine nucleotide-binding site and being the target for ADP-ribosylation catalyzed by pertussis toxin; toxin-catalyzed ADP-ribosylation uncouples the Go(alpha) protein from receptor and inhibits signaling. The signaling process depends on interaction of Go(alpha) with other components of the system, including hormone receptors, beta-gamma subunits, guanine nucleotides, and effector enzymes. To study the role of the various domains of Go(alpha), oligonucleotide-directed in vitro mutagenesis was used; mutants of Go(alpha) were expressed in E. coli, and partially purified. The recombinant Go(alpha) mutants were analyzed with regard to immunoreactivity with anti-Go(alpha) polyclonal antibodies and their ability to serve as substrates in the ADP-ribosylation reaction. Previously it was shown that mutants in which cysteine-351 was replaced with glycine, or in which two carboxy-terminal amino acid residues were deleted, were not substrates in the toxin-catalyzed ADP-ribosylation reaction. Deletion of the terminal tyrosine did not block ADP-ribosylation; replacement of leucine-357 with glycine or alanine produced mutants which were poor substrates for toxin-catalyzed ADPribosylation. These studies indicate that pertussis toxin recognizes the carboxy terminus of the alpha-chain; small differences in structure alter the ability of the mutant a chains to serve as ADP-ribose acceptors.